PROJECT SUMMARY Loss of homeostatic capacity is a fundamental and defining property of aged organisms from yeast to humans. The global protein kinase-substrate network forms an essential backbone of the homeostatic signaling network (HSN) that allows cells to respond appropriately to a dynamic environment and cellular needs. We propose that aging can be modeled as a series of changes to the HSN that directly impinge on core cellular functions. The overarching goal of this proposal is to model the HSN in yeast, and to understand the mechanisms by which degradation of the HSN results in functional declines and increasing risk of mortality with age. To accomplish this goal we will use a combination of global and targeted mass spectrometry approaches to map at high resolution the kinases and substrates that comprise the HSN. We will then expose aging yeast to perturbations, and measure changes in signaling using phosphoproteomics. Finally, we will combine these population level analyses with single-cell microfluidics measurements in order to define the penetrance and temporal dynamics of key components of the network (kinases and substrates) and reporters of cellular functions that are most prone to degradation throughout aging. This approach will allow us to determine, for the first time, the extent to which individual cells experience distinct aging trajectories. We will use this information to develop models for network degradation with age and to predict key components of the network prone to failure, which could potentially be strengthened to build a more robust network. We will test these predicted improvements by engineering them within yeast strains and assessing whether the strains indeed maintain important network structures with age, keep cellular functions of their youthful state, and, perhaps, live longer.